Abstract: The incidence and mortality rates for melanoma have been increasing for the last 30 years, with metastatic melanoma harboring a 5-year survival rate between 5 and 10%. These tumors develop mechanisms to avoid immune surveillance, including upregulation of inhibitory checkpoint ligands, increased production of immune-modulating cytokines, and decreased antigen expression. The advent of immunotherapies, drugs that overcome these immunosuppressive mechanisms such as anti-PD1, has offered substantial promise to improve patient outcomes. Unfortunately, variable response rates and the possibility for severe autoimmune reactions have hindered the efficacy of immunotherapies, necessitating identification of adjuvant therapeutic modalities. Focused Ultrasound (FUS), a non-invasive tissue ablation technology, has substantial potential to meet this need. Recent studies showing FUS enhances the immunogenicity of the tumor microenvironment (TME) have led to hypotheses that FUS could convert tumors from immunologically ?cold? to ?hot?, rendering them responsive to immunotherapy. Currently the therapeutic potential for FUS-immunotherapy combination remains unrealized due to incomplete i) mechanistic knowledge of FUS immunogenicity, ii) appreciation of compensatory resistance to FUS, and iii) optimization of the immunostimulatory profile induced in the TME. To overcome these barriers, high throughput transcriptomic and single cell methods will be performed in murine melanoma at multiple times post-FUS. These studies will be performed in the context of single or multiple FUS application to test whether repeated treatment, an understudied strength of FUS over radiation, can be leveraged to optimize immunogenicity. Insight gained from these studies will permit both selection of a FUS regimen that is maximally immunogenic and an immunotherapeutic with which to combine it, based explicitly on identified mechanisms of immunomodulation. The ability of selected FUS-immunotherapy combination strategies to provide primary and systemic tumor growth control will be systematically evaluated in two transplantable murine melanoma models, both of which are insensitive to checkpoint blockade alone. This represents an important step in leveraging FUS to sensitize refractory melanoma to immunotherapy. This proposal involves scientific, professional, and clinical training. The PI will complete formal coursework in cancer immunotherapy and receive direct guidance from immunologist collaborators at weekly meetings. The PI plans to communicate important findings at national and international conferences and in journal manuscripts. Finally, the PI will frequently shadow a clinical oncologist and maintain clinical skills. Research will be carried out under the guidance of therapeutic ultrasound expert, Dr. Richard Price at the University of Virginia, a highly cooperative environment that inspires patient-centric research. Indeed, the proposed work will be done in collaboration with oncologist Dr. Timothy Bullock, and clinicians Dr. Patrick Dillon and Dr. Craig Slingluff who are leading the world?s first FUS-immunotherapy clinical trials at UVa.